Beyond the Semen Analysis

For the man with an abnormal semen analysis, additional tests which may be recommended include specialized sperm tests, blood tests; and testis biopsy.

Antisperm Antibodies Test

The role of antisperm antibodies in causing male infertility is controversial, since no one is sure how common or how serious this problem is. However, some men (or their wives) will possess antibodies against the sperm, which immobilize or kill them and prevent them from swimming up towards the egg. The presence of these antibodies can be tested in the blood of both partners, in the cervical mucus, and in the seminal fluid. However, there is little correlation between circulating antibodies (in the blood) and sperm-bound antibodies (in the semen), There are many methods of performing this test, which can be quite difficult to standardize, as a result of which there is a lot of variability between the reports of different laboratories. The older methods of testing used agglutination methods on slides and in test tubes. Perhaps, the best method available today is one which uses immunobeads, which allow determination of the location of the antibodies on the sperm surface. If they are present on the sperm head they can interfere with the sperm's ability to penetrate the egg; if they are present on the tail they can retard sperm motility. Of course, if the test is negative, this is reassuring; the problem really arises when the test is positive! What this signifies and what to do about it are highly vexatious issues in medicine today, and doctors are even more confused about this aspect than patients.

Semen Culture Test

In the semen culture test, the semen sample is tested for the presence of bacteria, and , if present, their sensitivity to antibiotics is determined. Interpreting this test can also be problematic! It is normal to find some bacteria in normal semen samples - and the question which must be answered is : are these bacteria disease- causing or not?

Tests which assess the sperm's ability "to perform" include the following sperm function tests.

Postcoital Test (PCT)

The postcoital test is the easiest test of sperm function, since it is performed in vivo. It is done when the wife is in the " fertile" period, during which time the cervical mucus is profuse and clear. The gynecologist examines a small sample of the cervical mucus, under the microscope, a few hours after intercourse. (This can be embarrassing and awkward for the patient, but it is not painful at all). Finding 5-10 motile sperm per high power microscopic field means that the test is normal. A normal test implies normal sperm function and can be very reassuring.

An abnormal test needs to be repeated and, if the problem is persistent, one needs to determine if the defect lies in the sperm or in the mucus, by cross-testing with the husband's sperm, donor sperm, wife's mucus and donor mucus.

Bovine Cervical Mucus Test

The bovine cervical mucus test is another form of testing for the ability of the sperm to penetrate and swim through cervical mucus, with the difference that, in this case, the mucus used is that of a cow (since this is commercially available in a test kit.) The sperm are placed in a column of cervical mucus and how far the sperm can swim forward through the column in a given amount of time is checked with the help of a microscope.

Sperm Viability or Sperm Survival Test

This is a simple test, which provides crude (but useful!) information on the functional potential of the sperm. The sperm are washed using the same method which is used for IVF (either a Percoll spin or sperm swim up) and the washed sperm are then kept in a culture medium in the laboratory incubator for 24 hours. After 24 hours, the sperm are checked under the microscope. If the sperm are still swimming actively, this means that they have the ability to "survive" in vitro for this period and this is reassuring. If, however, none of the sperm are alive after 24 hours, this suggest that they may be functionally incompetent.

Sperm Penetration Assay (SPA, Hamster Assay)

Since the basic function of a sperm is to fertilize an egg, scientists were very excited when they found that normal sperm could penetrate a denuded (zona-free) hamster egg. A hamster egg is obtained from hamsters and the covering (the zone) removed by using special chemicals. The egg are then incubated with the sperm in an incubator in the laboratory. After 24 hours, the eggs are checked to ascertain how many sperm have been able to penetrate the egg. The result gives a penetration score, which gives an index of the sperm's fertilizing potential. This is a very delicate technique and is not available in India. In any case, nowadays scientists the world over are quite disenchanted with the test, since the correlation between IVF results (the ability to fertilize human eggs) and the SPA (the ability to penetrate zona-free hamster eggs) is quite poor.

Other specialized semen tests include:

Testing for acrosomal status.
HOS test hypo-osmotic swelling test which tests for the integrity of the sperm membrane.
CASA computer assisted sperm analysis.
Hemizona assay.
Electron microscopy of sperm.
The aforementioned tests are highly sophisticated and are not easily available. Another drawback is that these tests are often not standardized adequately, so that interpreting their results can be quite difficult.

The ultimate sperm function test is IVF, since this directly assesses whether or not the husband's" sperm can fertilize the wife's eggs. The best way to perform this test is to culture some of the eggs with the husband's sperm and the others with donor sperm of proven fertility, at the same time. If the donor sperm can fertilize the eggs, and the husband's sperm fail to do so, then the diagnosis of sperm inability to fertilize the eggs is confirmed. However, even this test is not infallible since it has been shown that about 5% of sperm samples which fail to fertilize an egg in the first IVF attempt, can do so in a second attempt at IVF. In any case, it is obviously not practicable or feasible to use IVF as a test for sperm function in clinical practice.

Blood Tests for Men

The serum FSH (follicle-stimulating hormone) level test is a very useful one for assessing testicular function. If the reason for the azoospermia or severe oligospermia is testicular failure, then this is reflected in a raised FSH level. This is because, in these patients, the testis also fails to produce a hormone called inhibin (which normally suppresses FSH levels to their normal range). A high FSH level is usually diagnostic of testicular failure. This test is done by a radioimmunoassay or ELISA test, and since it is a laboratary sophisticated test, it is best done in a specialized. Abnormal test results should be repeated and rechecked for confirmation. The other reason for a high FSH level in some men is the consumption of clomiphene (a medicine often prescribed for the empiric treatment of oligospermia). This is why the test should be done only when no medication is being taken. While a high FSH level is diagnostic of testicular failure, a normal FSH level provides no useful information. Rarely, the FSH level may be low. A low FSH level is found in patients with hypogonadotropic hypogonadism.

This is an uncommon (but treatable!) cause of azoospermia. Along with an FSH level test, most doctors also do an replacement therapy (in the form of injections or tablets). LH (luteinizing hormone) level test, which provides mostly the same information.

A testosterone level test provides information on whether or not the testes are producing adequate amounts of the male hormone, namely, testosterone. Most infertile men have normal testosterone levels, because the compartment for testosterone production is separate from the compartment which produces sperm, and is usually intact in infertile men. A low testosterone level causes a decreased libido and this can be treated by testosterone. Of course, this therapy will not increase the sperm count.

Ultrasound

An ultrasound of the testis has become a popular test to order, but its helpfulness is limited. The size of the testis is better assessed by clinical examination, using an orchidometer; and while a Doppler ultrasound will often diagnose the presence of a varicocele, this is usually of little clinical significance. The danger of finding a varicocele is that the knee-jerk response is to do surgery to correct it and this rarely benefits the patient. A transrectal ultrasound (TRUS) can be useful, but only in evaluating selected patients with obstructive azoospermia, when a block at the level of the seminal vesicles is suspected, and this test is best ordered by a specialist.

Testicular Biopsy

A testicular biopsy is done in order to find out whether sperm production in the testis is normal or not. This is the "gold standard" for judging testicular function, since here the testicular tissue is being examined directly. How is a testicular biopsy performed? This is a simple surgical procedure, which can be done under a local anaesthetic, in an operation theatre or even in the doctor's clinic, if it is well equipped. The test takes about 5-10 minutes to be carried out; and a biopsy could be taken from just one testis, or from both testes, depending upon the nature of the problem.

The removed bit of tissue is then placed in a special preservative fluid, which is then sent to a pathologist for examination under a microscope after staining.

The biopsy surgery doesn't hurt, because the local anesthetic numbs the tissues. There may be dull ache for a few days after the procedure, but this can be relieved by mild analgesics.

Since testis biopsy is a surgical procedure, most doctors would use it as the last resort when testing the man. If you are advised to have a testis biopsy, ask the doctor how the result will change your treatment (a question you should ask before being subjected to any medical test, in fact!).

The only group of infertile men who should be offered a testis biopsy are those with azoospermia. Men with oligospermia should not be subjected to a testis biopsy because the biopsy report is always normal in these men (and this is not surprising - after all, since sperm are present in the semen, they are obviously being produced in the testes!)

Formerly, when doctors performed a testis biopsy, they would send only one chunk of tissue for testing. However, today we know that a single biopsy may not be representative of the entire testis. Sperm production is not uniformly distributed throughout the testis, especially in men with testicular failure. This means that in order to get a true picture of sperm production in the testis, the doctor needs to sample at least 4 different areas of the testis, all of which need to be examined.

In the past, a testis biopsy was purely a diagnostic procedure. Today, it is also used to retrieve testicular sperm in order to treat men with severe male factor infertility. These testicular sperm can be used for intracytoplasmic sperm injection (ICSI), a procedure described in detail in Chapter. Specialised infertility clinics also have the ability to freeze the testicular tissue. This testicular sperm freezing can be very useful, especially in men with small testes, as the biopsy does not need to be repeated again during treatment.

The interpretation

While the biopsy is an easy test to perform, it is difficult to interpret properly, unless done by an expert. The doctor looks for evidence of sperm production in the seminiferous tubules. In some cases, there is no sperm production at all (absent spermatogenesis); or the sperm production is arrested at a particular stage (maturation arrest). This implies testicular failure, which is usually irreversible, and there is no treatment for this malady. If, on the other hand, sperm production in the testes is completely normal, and yet there are no sperm in the ejaculated semen, this clearly means that there is a block in the male reproductive tract. This is the one condition in which a testis biopsy is extremely useful (i.e., in the evaluation of the azoospermic male, to determine if there is a block to sperm transport).

A testis biopsy is often a procedure which is done badly because it is so "minor" so beware! It is preferable that the biopsy be done by a specialist; a poorly done biopsy may make reconstructive surgery on the epididymis more difficult later on, by causing adhesions and fibrosis (scarring). The commonest problem with the biopsy, however, is that the biopsy result is not reported accurately by the pathologist. Interpreting a testis biopsy is difficult and requires special expertise and is not something that the ordinary pathologist does well. You should retrieve and retain your own slides and preserve them carefully. The pathology laboratory can also be instructed to keep the tissue ("blocks") carefully. It is unfortunately common to find that a testis biopsy has to be repeated simply because the first one was done so badly that its results could not be accurately interpreted. It may also be a good idea to get a second specialist's opinion on the testis biopsy slides.

Vasography is another surgical test in which a radio- opaque dye is injected into the vas to determine if it is open, and, if blocked, to find out the exact site of the block. This test requires very delicate surgery and X-ray equipment and is a very infrequently done procedure because it can damage the vas.

For some men with testicular failure, a karyotype (study of the chromosomes) is useful, because it allows one to determine if a chromosomal problem (e.g., Klinefelter's syndrome, 47, XXY, with an extra X Chromosome) is responsible for the azoospermia. Some research clinics also offer testing for microdeletions on the Y-chromosome a newly discovered cause for testicular failure in about 15% of infertile men. While there is no treatment for this disorder, at least the test result provides an answer to the question of why the testes have failed a question which, unfortunately, medicine today still cannot answer, in the majority of patients.

Credits: Dr. Aniruddha Malpani, MD and DR. Anjali Malpani,